Method and apparatus for deriving oxygen association rate curves for blood samples

ABSTRACT

A method and apparatus for deriving an oxygen association .[.rate.]. curve for a blood sample wherein the sample is introduced into a transparent-walled cell and is deoxygenated by contact with a deoxygenated fluorocarbon material. The cell is located in the optical path of two time-shared respective monochromatic beams, one having a wavelength at which there is substantially no change in absorbance as between oxygenated and deoxygenated blood and the other having a wavelength at which there is a relatively large change in absorbance as between oxygenated and deoxygenated blood. The difference in absorbance through the cell of the two wavelengths is measured and recorded while oxygenated fluorocarbon titrant material is pumped through the cell, the cell having a restricted flow passage for the titrant material which acts to separate the blood therefrom and retain it in the cell while the titration is taking place.

This invention relates to methods and apparatus for measuring theoxygenation characteristics of blood or other material whoselight-absorbing characteristics change while being treated with areagent, and more particularly to a method and apparatus for deriving anoxygen association .[.rate.]. curve for a blood sample.

A main object of the invention is to provide a novel and improvedtechnique for differentially studying the light absorbing properties ofmaterials being treated with reagents which modify said light absorbingproperties, and particularly for measuring the .Iadd.degree or.Iaddend.rate of oxygenation of a blood sample while it is being treatedwith .Iadd.changing concentrations of .Iaddend.an oxygenating reagent.

A further object of the invention is to provide an improved method andapparatus for measuring and recording the change in light absorbance ofa blood sample being treated with an oxygenating reagent, the methodinvolving relatively simple procedural steps and requiring only a smallamount of the sample, and the apparatus being relatively inexpensive toconstruct and being easy to operate.

A still further object of the invention is to provide an improvedabsorbance cuvette or cell for use with a dual wavelength photometerapparatus for measuring the oxygenation characteristics of blood orsimilar material being treated with a reagent which changes thelight-absorbing characteristics of the material.

A still further object of the invention is to provide an improvedabsorbance cell for use with a dual wavelength photometer or similaroptical instrument for deriving the oxygen association .[.rate.]. curveof a blood sample, the cell enabling a sample to be readily injectedtherein and to be subsequently titrated with a liquid oxygenatingmaterial while time-shared light beams of specific wavelengths arepassed through the cell, enabling the differential absorbance of saidwavelengths to be measured while the sample is being oxygenated, thecell enabling the titrant to be pumped through the cell while retainingthe blood sample therein.

Further objects and advantages of the invention will become apparentfrom the following description and claims, and from the accompanyingdrawings, wherein:

FIG. 1 is an elevational view, partly in vertical cross-section, of animproved absorbance-measuring cell adapted to be employed with a dualwavelength photometer in accordance with the present invention.

FIG. 2 is a horizontal cross-sectional view taken substantially on theline 2--2 of FIG. 1.

FIG. 3 is an enlarged fragmentary vertical cross-sectional view takenthrough the phase separation, or blood retention, portion of the cell ofFIGS. 1 and 2.

FIG. 4 is an enlarged fragmentary horizontal cross-sectional view takensubstantially on the line 4--4 of FIG. 3.

Reference is made to the previously filed patent application of GeorgeW. Lowy, Paul Priarone and Herbert M. Cullis, Ser. No. 291,046, filedSept. 21, 1972, now issued as U.S. Pat. No. 3,787,124, entitled "DualWavelength Photometer for Absorbance Difference Measurements." Thispreviously filed application discloses a dual wavelength photometerwherein timeshared monochromatic wavelengths λ_(R) and λ_(M),respectively 448 nm and 439 nm, are sequentially passed through acuvette containing a blood sample being titrated by an oxygenatingmaterial, the beams impinging on a photomultiplier tube, and appropriatecircuitry being provided to segregate the responses produced by the twowavelengths and to record the absorbance difference of the blood sampleduring the course of the oxygenating titration. As is pointed out insaid previously .[.filled.]. .Iadd.filed .Iaddend.application, thereference wavelength λ_(R), namely, 439 nm is of a value at which thereis a relatively large change in absorbance as between oxygenated anddeoxygenated blood. The instrument thus plots the difference inabsorbance for the two wavelengths with time as the sample is beingoxygenated.

In the apparatus disclosed in said previously filed application, oxygenis generated at a constant rate in an optical cell containing a bloodsample in the liquid phase, employing a dilute hydrogen peroxidesolution of known concentration as titrant and catalese as adecomposition catalyst. Oxygen is thereby generated stoichiometricallyat a rate which is proportional to the rate .Iadd.(incremental orcontinuous change in concentration) .Iaddend.of the controlled titrantflow into the curvette.

Improved results can be obtained by employing a liquid perfluorinatedcarbon compound as the titrant, for example, Mediflor FC-43, FC-47 orFC-80, manufactured by the 3M Company, Inc., Minneapolis, Minnesota,which are fluorocarbon materials which can be readily deoxygenated andoxygenated, and which can be efficiently used as deoxygenating andoxygenating reagents for blood samples. One of the objectives of thepresent invention therefore is to provide a cuvette wherein this type oftitrant can be satisfactorily employed and which can be incorporated foruse in a dual wavelength photometer of the type described in saidpreviously filed application, or in other absorbance-measuringinstruments.

Referring now to the drawings, 11 generally designates an improvedtitration cell for use in an oxygen association rate curve-derivingtechnique according to the present invention. The typical titration cell11 comprises a metal block 12 of generally rectangular shape providedwith a plurality of mounting apertures 13. The block is formed with acentral cavity 14 and is provided on opposite sides of the cavity withtransparent parallel circular window discs 15, 15 clampingly secured incircular recesses provided therefor by retaining rings 16, 16 threadedlyengaged in the circular recesses. Flat annular sealing gaskets 17, 17are provided between the clamping rings 16 and the transparent windowdiscs 15.

The block 12 is formed at one side thereof with a large rectangularnotch 18, and a flanged injection guide conduit member 19 is threadedlyengaged in a bore 20 extending from the midportion of the notch 18toward the cavity 14. The inner end of the bore 20 adjacent cavity 14 isreduced to define an annular retaining seat 21 against which a rubberseptum disc 22 is clamped by the inner end of conduit member 19. Theseptum disc 22 is penetrable by the hollow needle 23 of a hypodermicsyringe 24 employed to inject a blood sample into cavity 14, as will bepresently described.

The block 12 is formed with drilled passages 25 extending around cavity14 and appropriately plugged at ends thereof to define a means forcirculating temperature-controlling liquid so as to maintain thetemperature of the block at a controlled value. The temperature-controlliquid is admitted to the passages through an inlet conduit 26, and theliquid leaves the block through an outlet passage 27, whereby the liquidcan circulate through the passages 25 and thereby maintain a constanttemperature in the cavity 14.

As will be presently explained, it is desirable to agitate the contentsof cavity 14, and therefore an agitating mechanism 28 is provided on thetop portion of block 12, said mechanism being driven by a rotary shaft29 journalled in a bearing bar 30 suitably secured to the main block 12.The shaft 29 reciprocates an agitator rod 31 extending into cavity 14,said rod having spaced agitating flanges 32. The mechanism 28 isprovided with a conventional motion-converting assembly 33 between rod31 and shaft 29 which converts the rotary movement of the shaft intoreciprocating axial movement of rod 31.

The agitator rod 31 extends into cavity 14 through a housing sleeve 49mounted in the upper portion of block 12 and provided at its bottom endwith a flexible cover diaphragm 50, the rod being sealingly secured toand extending through the center portion of said diaphragm.

Block 12 is formed with a counterbored bottom filling passage 34 inwhich is seated a sleeve member 35 having a lower central bore portion36 in which is secured the end of a filling tube 37. A screw bushing 38surrounds tube 37 and is threadedly engaged with the block 12 belowsleeve member 35 and clamps said sleeve member against a resilientdeformable O-ring 39 engaged around the reduced top neck portion 40 ofsleeve member 38, the O-ring 39 being thus forced against the annularseat 41 defined at the top end of counterbored passage 34.

Sleeve member 38 has an enlarged vertical upper bore portion 42communicating with cavity 14. A vertical outlet tube 43 extends throughthe top portion of block 12 in alignment with bore portion 42 and isprovided at its lower end with an elongated collar member 44, said tubelower end and collar member extending downwardly into bore portion 42 inthe manner shown in FIG. 3 and defining a generally cylindricalconstricted passage 45 between cavity 14 and the lower end of outlettube 43. The spacing between the exterior surface of collar member 44and bore portion 42 is of the order of 0.005 inch and the height of thecollar member in said bore portion is substantial, being of the order ofat least 1/2 inch, so that the constriction 45 has substantial axiallength. As will be presently explained, the axial length of saidconstriction is sufficient to enable blood to separate from reagentliquid flowing from cavity 14 downwardly into bore portion 42 toward thebottom end of outlet tube 43 while the mixture in cavity 14 is beingagitated by the action of rod 31.

A flushing tube 46 is secured in the top portion of block 12 incommunication with the top of cavity 14 for a purpose presently to bedescribed.

A titrant admission tube 51 extending to and communicating with theupper corner portion of cavity 14 opposite conduit member 19 is securedin block 12. Tube 51 is connected to a suitable syringe pump containingoxygenated fluorocarbon material, such as Mediflor FC-43, or the like,as above identified.

Input shaft 29 of agitator driving mechanism 28 is connected to asuitable driving means, such as an electric motor or the like, notshown.

The cell 11 is mounted in the optical path of the associated dualwavelength photometer so that the timeshared λ_(R) and λ_(M) light beamspass transversely through the windows 15 and substantially through anarea shown in dotted view at 47.

In operating the apparatus, a quantity of fluorocarbon liquid material,such as Mediflor FC-43, or the like, is first deoxygenated by externalconventional means, such as degassing the nitrogen or other inert gas,and is inserted into cavity 14 through filling tube 37, completelyfilling the cavity, overflow taking place through the exit tube 43.

With the cell 11 mounted in the photometer, a blood sample (typicallypartially oxygenated) is then injected into the cavity 14 through theconduit member 19 by means of a hypodermic syringe, as illustrated indotted view in FIG. 2.

The agitator mechanism 28 is activated and remains in operationthroughout the remaining procedure.

At this stage, deoxidation of the injected blood sample takes place bycontact with the fluorocarbon liquid and because of dilution. Completedeoxidation of the blood sample is then performed by flushing the cavity14 with deoxygenated fluorocarbon material admitted into the cavitythrough the flushing tube 46.

The optical system is then balanced by employing a procedure similar tothat described in previously filed application Ser. No. 291,046, aboveidentified, and the electronic system thereof is activated so that theinstrument begins to record.

Externally oxygenated fluorocarbon material (Mediflor FC-43, or thelike) is then pumped into the cavity 14 through tube 51, which beginsthe titration process. In this step, and in the previously describedflushing (deoxidation of the blood sample) step, excess fluorocarbonmaterial leaves the cell through the "phase separator" restriction 45and tube 43, the blood being retained because the blood floats back upinto cavity 14 while the relatively less bouyant fluorocarbon materialpasses through the restriction 45 and is flushed out through tube 43.The optical measurements are made during this stage of the procedure andan oxygen association .Iadd.degree or .Iaddend.rate curve is recorded inan manner similar to that described in the previously filed applicationSer. No. 291,046.

While a specific embodiment of an improved method and apparatus forderiving an oxygen association .[.rate.]. curve for a blood sample hasbeen disclosed in the foregoing description, it will be understood thatvarious modifications within the spirit of the invention may occur tothose skilled in the art. Therefore it is intended that no limitationsbe placed on the invention except as defined by the scope of theappended claims.

What is claimed is:
 1. A method of deriving an oxygen association ratecurve for a blood sample comprising deoxygenating the sample,introducing an oxygenating agent into the sample, passing two respectivemonochromatic wavelengths through the sample while it is beingoxygenated, one wavelength at which there is substantially no change inabsorbance as between oxygenated and deoxygenated blood and the otherwavelength at which there is a relatively large change in absorbance asbetween oxygenated and deoxygenated blood, and plotting the differencein absorbance for the two wavelengths with time as the sample is beingoxygenated.
 2. The method of claim 1, and wherein the blood sample andthe oxygenating agent are agitated while oxygenation of the blood sampleis taking place.
 3. The method of claim 1, and wherein said onewavelength has a value of approximately 448 nm and said other wavelengthhas a value of approximately 439 nm.
 4. The method of claim 1, andwherein the oxygenating agent comprises oxygenated fluorocarbon liquidmaterial.
 5. The method of claim 4, and wherein deoxygenation of thesample comprises exposing it to deoxygenated fluorocarbon liquidmaterial. .Iadd.
 6. A method of deriving oxygen association curveinformation for a blood sample, said method comprising disposing a bloodsample within a treatment cell, introducing available oxygen into saidcell in a first concentration, varying said first concentration ofoxygen within said cell over a period of time to provide exposure ofsaid blood sample to varying concentrations of oxygen, passing first andsecond light beams through said sample during said time period, saidfirst light beam having a wavelength such that there is substantially nodifference in absorbance of said wavelength by said blood sample in theoxygenated and deoxygenated states, respectively, of said blood, saidsecond light beam having a wavelength such that there is a relativelylarge difference in absorbance of said wavelength by said blood samplein the oxygenated and deoxygenated states, respectively, of said blood,and determining the changes in oxygen association of said blood withrespect to the changes in oxygen concentration within said cell bysensing the differences in the respective degrees of absorption of saidfirst and second beams by said blood as said oxygen concentration ischanged. .Iaddend..Iadd.
 7. A method as defined in claim 6 whereinvarying said concentration of oxygen includes the steps of substantiallydeoxygenating said sample and thereafter introducing oxygenatingmaterials into said cell in gradually increasing concentrations untilsaid sample is substantially completely oxygenated. .Iaddend. .Iadd. 8.A method as defined in claim 6 wherein said one wavelength has a valueof approximately 448 nm and said other wavelength has a value ofapproximately 439 nm. .Iaddend..Iadd.
 9. A method as defined in claim 1wherein said available oxygen is introduced in the form of anoxygenating agent, and wherein the blood sample and said oxygenatingagent are agitated while oxygenation of the blood sample is takingplace. .Iaddend..Iadd.
 10. A method as defined in claim 1 wherein saidavailable oxygen is introduced in the form of an oxygenating agent, andwherein the oxygenating agent comprises oxygenating flurocarbonliquidated material. .Iaddend..Iadd.
 11. A method as defined in claim 1which further includes the step of deoxygenating said sample prior tooxygenating said sample, said deoxygenating being accomplished byexposing said sample to deoxygenating fluorocarbon liquidated material..Iaddend. .Iadd.
 12. A method of deriving oxygen association curveinformation for a blood sample, said method comprising disposing a bloodsample within a treatment cell, introducing available oxygen into saidcell in a first concentration, varying said first concentration ofoxygen within said cell over a period of time to provide exposure ofsaid blood sample to varying concentrations of oxygen, passing first andsecond light beams through said sample during said time period, saidfirst light beam having a first wavelength selected so as to have afirst characteristic difference in absorbance thereof by said bloodsample as between the oxygenated and deoxygenated states thereof, saidsecond light beam having a second wavelength selected so as to have asecond characteristic difference in absorbance of said wavelength bysaid blood sample as between the oxygenated and deoxygenated statesthereof, and determining the changes in oxygen association of said bloodwith respect to the changes in oxygen concentration within said cell bysensing the differences in the respective degrees of absorption of saidfirst and second beams by said blood as said oxygen concentration ischanged. .Iaddend..Iadd.
 13. A method as defined in claim 12 whereinvarying said concentration of oxygen includes the steps of substantiallydeoxygenating said sample and thereafter introducing oxygenatingmaterials into said cell in gradually increasing concentrations untilsaid sample is substantially completely oxygenated. .Iaddend..Iadd. 14.A method as defined in claim 12 wherein said first wavelength has avalue of approximately 448 nm and said second wavelength has a value ofapproximately 439 nm. .Iaddend. .Iadd.
 15. A method of deriving oxygenassociation curve information for a blood sample, said method comprisingdisposing a blood sample within a treatment cell, introducing availableoxygen into said cell in a first concentration, varying said firstconcentration of oxygen within said cell over a period of time toprovide exposure of said blood sample to varying concentrations ofoxygen, passing first and second light beams through said sample duringsaid time period, said first light beam having a first wavelengthselected so as to have a first, characteristic difference in absorbancethereof by said blood sample as between the oxygenated and deoxygenatedstates, respectively, of said blood, said second light beam having asecond wavelength selected so as to have a second characteristicdifference in absorbance of said wavelength by said blood sample asbetween the oxygenated and deoxygenated states, respectively, of saidblood, and determining the changes in oxygen association of said bloodwith respect to the changes in oxygen concentration within said cell bysensing the differences in the respective degrees of absorption of saidfirst and second beams by said blood as said oxygen concentration ischanged. .Iaddend..Iadd.
 16. A method as defined in claim 15 whereinvarying said concentration of oxygen includes the steps of substantiallydeoxygenating said sample and thereafter introducing oxygenatingmaterials into said cell in gradually increasing concentrations untilsaid sample is substantially completely oxygenated. .Iaddend. .Iadd. 17.A method as defined in claim 15 wherein said first wavelength has avalue of approximately 448 nm and said second wavelength has a value ofapproximately 439 nm. .Iaddend.